Microbial pathogens greatly reduce yields of a variety of important food crops (e.g., corn, rice), threaten entire industries (e.g., rubber, tobacco), and devastate ornamental plants and trees (e.g., elm, chestnut, ash). Unfortunately, treatment with broad-spectrum antimicrobial agents destroys, in addition to the pathogen, important commensal or non-pathogenic organisms and, thus, can facilitate the subsequent colonization by additional pathogens. Therefore, the ideal antimicrobial treatment or therapy is a substance that selectively kills or eliminates specific pathogenic organisms while having minimal effects on the microbial ecology. The importance of the microbial ecology is well-illustrated in the successful use of xe2x80x9cantagonistic yeasts,xe2x80x9d such as Epicoccum nigrum, Penicillium oxalicum, and Candida sake, in the control of brown rot of peaches and other fruit. In these examples, protection is a result of the ability of the applied fungal strains to rapidly colonize the fruit and prevent subsequent colonization by organisms associated with post-harvest decay. While these antagonistic approaches can serve as prophylactic treatments, they are much less effective displacing or selectively killing pathogenic or undesirable microorganisms once colonization has occurred.
Selective killing of a microorganism can be achieved by development of a substance that is either selectively toxic or one that is generally toxic but selectively targeted. Development of either type of antimicrobial agent is plagued by the inherent similarities between pathogenic and non-pathogenic organisms at the level of physiology, nutrient requirements, and biology.
Virtually all organisms mate or fuse to allow exchange of genetic and/or other intracellular components. In fungi, fusion is not a random event, but rather is restricted to occur only between members of the same species, and often includes a further dependency on secondary characteristics such as mating type and vegetative compatibility group (VCG). For instance, haploid strains of S. cerevisiae will mate and fuse only if they are of opposite mating types. In addition to mating, many filamentous fungi are also able to undergo anastomosis (hyphal fusion) but only if they are of the same vegetative compatibility group. Importantly, these fusion reactions occur with absolute selectivity.
We have harnessed the biological discriminatory mechanisms described above to selectively target and kill pathogens.
Accordingly, in a first aspect, the invention features a method of selectively killing a first microorganism. The method includes: (i) contacting the first microorganism with a second microorganism that contains a microcidal compound; and (ii) allowing the first microorganism and the second microorganism to undergo fusion, whereby the microcidal compound is delivered into and kills the microorganism that forms following the fusion.
In a preferred embodiment, the first microorganism is fungus. Preferred fungi include Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria boydii, Altemaria spp., Anthopsis deltoidea, Aphanomyces spp., Apophysomyces eleqans, Armillaria spp., Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Botrytis spp., Candida spp., Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis, Colletotrichum spp, Conidiobolus spp., Corynebacterium tenuis, Cryptoporiopsis spp., Cylindrocladium spp., Cryptococcus spp., Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp., Diplodia spp., Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fulvia spp., Fusarium spp., Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Macrophomina spp., Madurella spp., Magnaporthe spp., Malasseziafurfur, Microsporum spp., Monilinia spp., Mucor spp., Mycocentrospora acerina, Nectria spp., Nocardia spp., Oospora spp., Ophiobolus spp., Paecilomyces spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phlyctaena spp., Phoma spp., Phomopsis spp., Phymatotrichum spp., Phytophthora spp., Pythium spp., Piedraia hortai, Pneumocystis carinii, Puccinia spp., Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizoctonia spp., Rhizopus spp., Saccharomyces spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Scerotium spp., Sclerotinia spp., Sphaerotheca spp., Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Taphrina spp., Thielaviopsis spp., Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Ustilago spp., Venturia spp., Verticillium spp., Wangiella dermatitidis, Whetxelinia spp., Xylohypha spp., and their synonyms.
In another preferred embodiment, the compound is a toxic compound or is a compound that causes a toxic compound to be produced in the microorganism that forms following fusion. Preferably, the toxic compound is a toxin or fragment thereof selected from the group consisting of: diphtheria toxin, diphtheria toxin F2 fragment, diphtheria toxin A domain, Pseudomonas exotoxin A, and the A domain of Pseudomonas exotoxin A. Alternatively, the compound is a biosynthetic enzyme that causes a toxic compound to be produced in the microorganism that forms following fusion.
In a related embodiment, the second microorganism is resistant to the microcidal compound. In preferred embodiments, the second microorganism is a nonpathogenic fungus.
In a second aspect, the invention features a method for producing a diphtheria toxin-resistant fungus. The method includes introducing into the fungus a mutation in its DPH1, DPH3, or DPH4 gene that prevents the biosynthesis of diphthamide, wherein the fungus is selected from the group consisting of: Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria boydii, Altemaria spp., Anthopsis deltoidea, Aphanomyces spp., Apophysomyces eleqans, Armillaria spp., Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Botrytis spp., Candida spp., Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis, Colletotrichum spp, Conidiobolus spp., Corynebacterium tenuis, Cryptoporiopsis spp., Cylindrocladium spp., Cryptococcus spp., Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp., Diplodia spp., Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fulvia spp., Fusarium spp., Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Macrophomina spp., Madurella spp., Magnaporthe spp., Malassezia furfur, Microsporum spp., Monilinia spp., Mucor spp., Mycocentrospora acerina, Nectria spp., Nocardia spp., Oospora spp., Ophiobolus spp., Paecilomyces spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phlyctaena spp., Phoma spp., Phomopsis spp., Phymatotrichum spp., Phytophthora spp., Pythium spp., Piedraia hortai, Pneumocystis carinii, Puccinia spp., Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizoctonia spp., Rhizopus spp., Saccharomyces spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformnis, Scerotium spp., Sclerotinia spp., Sphaerotheca spp., Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Taphrina spp., Thielaviopsis spp., Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Ustilago spp., Venturia spp., Verticillium spp., Wangiella dermatitidis, Whetxelinia spp., and Xylohypha spp.
In a third aspect, the invention features a diphtheria toxin-resistant fungus containing a mutation in its DPH1, DPH3, or DPH4 gene that prevents the biosynthesis of diphthamide, wherein the fungus is selected from the group consisting of: Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria boydii, Alternaria spp., Anthopsis deltoidea, Aphanomyces spp., Apophysomyces eleqans, Armillaria spp., Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Botrytis spp., Candida spp., Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis, Colletotrichum spp, Conidiobolus spp., Corynebacterium tenuis, Cryptoporiopsis spp., Cylindrocladium spp., Cryptococcus spp., Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp., Diplodia spp., Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fulvia spp., Fusarium spp., Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Macrophomina spp., Madurella spp., Magnaporthe spp., Malassezia furfur, Microsporum spp., Monilinia spp., Mucor spp., Mycocentrospora acerina, Nectria spp., Nocardia spp., Oospora spp., Ophiobolus spp., Paecilomyces spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phlyctaena spp., Phoma spp., Phomopsis spp., Phymatotrichum spp., Phytophthora spp., Pythium spp., Piedraia hortai, Pneumocystis carinii, Puccinia spp., Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizoctonia spp., Rhizopus spp., Saccharomyces spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Scerotium spp., Sclerotinia spp., Sphaerotheca spp., Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Taphrina spp., Thielaviopsis spp., Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Ustilago spp., Venturia spp., Verticillium spp., Wangiella dermatitidis, Whetxelinia spp., and Xylohypha spp. In a preferred embodiment of the second or third aspect, the gene hybridizes at high stringency to S. cerevisiae DPH1, DPH3, or DPH4 and has DPH activity.
In a fourth aspect, the invention features a substantially pure preparation of a Dph3 polypeptide.
In preferred embodiments, the Dph3 polypeptide is at least 55% identical to the amino acid sequence of FIG. 7 (SEQ ID No: 10), is from a fungus (e.g., Saccharomyces cerevisiae), and has DPH biological activity.
In a fifth aspect, the invention features DNA encoding a Dph3 polypeptide.
In preferred embodiments, the DNA includes the DPH3 gene of FIG. 7 (SEQ ID No: 9) and complements a DPH3 mutation in Saccharomyces cerevisiae.
By xe2x80x9cfusionxe2x80x9d is meant any combination of two organisms or cells that leads to the exchange, mixing, or transfer of intracellular contents. Preferred forms of fusion are mating and anastomosis.
By xe2x80x9ckillxe2x80x9d is meant to induce death in the microorganism that forms following fusion, resulting in a reduction in the number of microorganisms. Preferably, the reduction is at least 25%; more preferably the reduction is 50%; and most preferably the reduction in the number of microorganisms is 75% or even 95%.
By a xe2x80x9cmicrocidal compoundxe2x80x9d is meant a molecule that leads to the induction of death of the microorganism. Exemplary microcidal compounds include, but are not limited to, molecules that are themselves toxic, molecules that induce production of a toxic compound, molecules that are toxic when combined with additional molecules, and DNA or RNA molecules encoding any of the foregoing molecules.
By xe2x80x9cDPH biological activityxe2x80x9d is meant an activity required for the biosynthesis of diphthamide; lack of said activity confers resistance to toxic compounds, including diphtheria toxin, diphtheria toxin F2 fragment, diphtheria toxin A domain, Pseudomonas exotoxin A, and the A domain of Pseudomonas exotoxin A.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By xe2x80x9chigh stringency conditionsxe2x80x9d is meant hybridization in 2xc3x97 SSC at 40xc2x0 C. with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley and Sons, New York, N.Y., 1994, hereby incorporated by reference.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide (for example, a polypeptide such as a Dph3 polypeptide) that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a Dph3 polypeptide. A substantially pure Dph3 polypeptide may be obtained, for example, by extraction from a natural source (for example, a fungal cell); by expression of a recombinant nucleic acid encoding a Dph3 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By xe2x80x9cisolated DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
This invention provides significant advances over existing methods of controlling pathogenic microorganisms.
First, in contrast to chemical agents, this method is highly selective; only the target microorganism is eliminated and, thus, protective elements of the microbial ecosystem are left intact.
Second, unlike the application of xe2x80x9cantagonisticxe2x80x9d biocontrols to control post-harvest disease, the method of the invention actively kills target organisms and, thus, is expected to be more successful in eliminating established infections.
Third, unlike the use of hypoviruses to attenuate pathogenic strains of Cryphonectria parasitica (the causative agent of chestnut blight), the method described herein is broadly adaptable and can utilize a variety of toxic compounds and strains. It is not restricted by the identification of a suitable virus or the limited host-range of the infectious agent.
Finally, because mutation of DPH1, DPH2, DPH4, or DPH5 does not significantly affect cell growth, the killer organism is expected to grow robustly and perform well in the environment.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.